ABSTRACT Induction of immune tolerance with solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. Induction of immune tolerance to mismatched grafts would obviate the need for life- long immunosuppression which is associated with serious adverse outcomes, such as renal failure, cancers and infections. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of tolerogenic organs such as kidneys, the mixed chimerism approach has not achieved durable immune tolerance in preclinical or clinical trials with most solid organs or vascular composite allotransplants (VCA). Encouragingly, though, we have succeeded in achieving reduced immunosuppression in clinical trials of VCA using this approach. Mesenchymal stem (stromal) cells (MSC) are a potentially useful adjuvant to stem cell transplants (SCT) for promoting mixed chimerism as well as promoting complementary peripheral immunomodulatory functions. However, there are many unresolved issues to address before clinical translation of these promising therapeutic cells. A primary impediment is the source of MSC, which are rare in all tissues and require invasive procedures for procurement. Low abundance mandates extensive expansion in culture to generate sufficient numbers for human dosing. It has been observed in the clinical setting that the degree of expansion is negatively correlated with outcomes. Ossium Health has overcome this obstacle by identifying an abundant source of primary MSC associated with medullary bones of vertebral bodies obtained from deceased organ donors. These vertebral bone adherent MSC (vBA-MSC) are isolated by proteolytic digestion of bone fragments, following elution and cryopreservation of bone marrow (BM). Primary vBA-MSC are obtained at numbers that are 3 orders of magnitude higher than can be recovered from living donor iliac crest BM aspirates. A further advantage of vBA-MSC is they are matched to the donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than BM-MSC, but, because of their high numbers, unlike traditional BM-MSC, can be expanded to >5 billion cells with only 2 passages in culture. We hypothesize that donor-matched vBA-MSC will augment tolerance mechanisms of mixed chimerism with BM transplant as well as provide peripheral immunomodulatory functions to achieve durable tolerance for major histocompatibility complex mismatched solid organ and vascular composite tissue transplants. This hypothesis will be tested first in a murine orthotopic hind limb transplant VCA model and then in a murine heterotopic heart model. The hindlimb model studies will allow us to evaluate mechanisms of MSC immune tolerance due to the tolerogenic nature of BM-containing hind limbs. Durable grafts will be evaluated for T cell dynamics (especially memory T cells and regulatory T cells) and donor-specific immune tolerance with be confirmed with donor and third-party skin grafting. Information gained from this study will be used to perform similar studies in our heterotopic heart transplant model. If successful, the results of this study, combined with a plethora of MSC clinical trials as well as a long history of transplantation tolerance trials and our future Phase II studies to further define dosing parameters in small and large animal models, will provide compelling arguments to FDA for proceeding to clinical trials. Keywords: vascular composite allotransplantation; solid organ transplantation; immune tolerance; immunomodulation, regulatory T lymphocytes